Bacteria have been used to produce a wide range of commercial products. For example, many Streptomyces strains and Bacillus strains have been used to produce antibiotics; Pseudomonas denitrificans and many Propionibacterium strains have been used to produce vitamin B12; some other bacteria have been used to produce vitamins such as Riboflavin; Brevibacterium flavum and Corynebacterium glutamicum have been used to produce lysine and glutamic acid, respectively, as food additives; other bacteria have been used to produce other amino acids used as food additives; Alcaligenes eutrophas has been used to produce biodegradable microbial plastics; and many Acetobacter and Gluconobacter strains have been used to produce vinegar. More recently, bacteria, such as Escherichia coli (E. coli), have been genetically engineered and used as host cells for the production of biological reagents, such as proteins and nucleic acids, in laboratory as well as industrial settings. The pharmaceutical industry supports several examples of successful products, which are human proteins manufactured in E. coli cultures cultivated in a fermenter.
E. coli K-12 is the most commonly used host for cloning and other molecular biology techniques and is the platform of choice for production of metabolites such as amino acids and many proteins of therapeutic or commercial interest. Recently it has been used or proposed for production of therapeutic DNA for use in gene therapy, DNA vaccines, and RNA interference applications. The complete genomes of two closely related E. coli K-12 strains, MG1655 and W3110, have been sequenced and are available from the National Center for Biotechnology Information microbial genomes database (NCBI database) (www.ncbi.nih.gov/genomes/lproks.cgi) as accession numbers U00096 and AP009048 respectively. Eighty-seven percent of E. coli K-12 genes have been assigned functions with some degree of confidence, making it one of the best understood organisms.
Desirable properties for a platform microorganism include efficiency of production, purity of product and stability of the genome during experimental manipulation, in production, and in storage. The chromosome of E. coli is littered with mobile genetic elements that mediate horizontal gene transfer, including insertion sequences (IS), transposases, defective phages, integrases, and site-specific recombinases. These elements can translocate, duplicate, and be maintained in the genome like an infectious agent, and are known to hop into plasmids as well. IS elements may cause inversions, duplications, and deletions mediated by homologous recombination. This can happen even when the transposase function has become inactive. Similar rearrangements also result from rRNA and Rhs repeats, but the instability is magnified when active transposases are involved.
Genome alterations due to IS translocation occur surprisingly frequently, and many commonly used laboratory and industrial strains have unrecognized genome alterations from this cause. For example, many of the differences between the two sequenced E. coli K-12 strains, which have been separated for about five decades from a common laboratory ancestor, are due to IS hops. The sequence databases provide ample evidence that IS hopping into plasmids is also common on the time scale of laboratory manipulations. Approximately one in every thousand eukaryotic sequences in the public databases is inadvertently contaminated with bacterial IS elements that apparently hopped into the cloned eukaryotic DNA during the brief period of propagation in E. coli prior to sequencing.
IS elements can also be inadvertently introduced into strains by laboratory manipulations. A case in point involves the E. coli K-12 derivatives DH10B and DH5α, which carry an IS10 not present in the ancestral K-12 genome. Despite a report that residual IS10 elements do not exhibit transpositional mutagenesis in recA strains, such as DH10B and DH5α, the prominence of IS10 contamination of the eukaryotic databases shows that this continues to be an issue. Thus, IS elements may lead to unpredictable consequences with important production hosts and pose a considerable impediment to the efficiency and accuracy of amino acid, protein, and nucleic acid production in E. coli. 